<div><p><i>Vibrio cholerae</i> is an aquatic gram-negative microbe responsible for cholera, a pandemic disease causing life-threatening diarrheal outbreaks in populations with limited access to health care. Like most pathogenic bacteria, <i>V</i>. <i>cholerae</i> secretes virulence factors to assist colonization of human hosts, several of which bind carbohydrate receptors found on cell-surfaces. Understanding how pathogenic virulence proteins specifically target host cells is important for the development of treatment strategies to fight bacterial infections. <i>Vibrio cholerae</i> cytolysin (VCC) is a secreted pore-forming toxin with a carboxy-terminal β-prism domain that targets complex <i>N</i>-glycans found on mammalian cell-surface proteins. To investigate glycan selectivity, we studied the VCC β-prism domain and two additional β-prism domains found within the <i>V</i>. <i>cholerae</i> biofilm matrix protein RbmC. We show that the two RbmC β-prism domains target a similar repertoire of complex <i>N</i>-glycan receptors as VCC and find through binding and modeling studies that a branched pentasaccharide core (GlcNAc<sub>2</sub>-Man<sub>3</sub>) represents the likely footprint interacting with these domains. To understand the structural basis of <i>V</i>. <i>cholerae</i> β-prism selectivity, we solved high-resolution crystal structures of fragments of the pentasaccharide core bound to one RbmC β-prism domain and conducted mutagenesis experiments on the VCC toxin. Our results highlight a common strategy for cell-targeting utilized by both toxin and biofilm matrix proteins in <i>Vibrio cholerae</i> and provide a structural framework for understanding the specificity for individual receptors. Our results suggest that a common strategy for disrupting carbohydrate interactions could affect multiple virulence factors produced by <i>V</i>. <i>cholerae</i>, as well as similar β-prism domains found in other vibrio pathogens.</p></div>